Recombinant hK2 polypeptide

ABSTRACT

An isolated, substantially homogenous pre-pro, pro or mature hK2 polypeptide is provided as well as biologically active variants or subunits thereof, including a biologically active variant pre-pro hK2 polypeptide having SEQ ID NO:19.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.08/427,767, filed May 2, 1995, now abandoned, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/241,174,filed May 10, 1994, now abandoned the disclosure of which isincorporated by reference herein.

BACKGROUND OF THE INVENTION

The glandular kallikreins are a subgroup of serine proteases which areinvolved in the post-translational processing of specific polypeptideprecursors to their biologically active forms. The human kallikrein genefamily consists of three members: prostate-specific antigen, humanglandular kallikrein, and pancreatic/renal kallikrein. See J. A.Clements, Endocr. Rev., 10, 393 (1989) and T. M. Chu et al. (U.S. Pat.No. 4,446,122). A common nomenclature for these members of the tissue(glandular) kallikrein gene families was recently adopted by T. Berg etal., in Recent Progress on Kinins: Biochemistry and Molecular Biology ofthe Kallikrein-Kinin System. Agents and Actions Supplements, Vol. I., H.Fritz et al.., eds., Birkkauser Verlag, Basel (1992), and is defined inTable I, below.

                  TABLE 1                                                         ______________________________________                                        The Human Tissue Kallikrein Gene Family                                        (approved species designation: HSA)                                            New      Previous                                                                                                   Designa- Designa-   New Protein                                               tion tions mRNA/cDNA Protein                                                 Designation                            ______________________________________                                        hKLK1  KLK1     HK1 and    tissue kalli-                                                                           hK1                                         hRKALL phKK25 krein (renal/                                                    cDNAs pancrease/sali-                                                          vary)                                                                      hKLK2 KLK2  prostate-specific hK2                                              hGK-1  glandular kalli-                                                       hKK-3  krein                                                                 hKLK3 PSA HPSA-1 PSA (prostate- hK3                                            PA and PSA specific antigen)                                                  APS cDNAs                                                                  ______________________________________                                    

The DNA sequence homology between hKLK2 and hKLK3 (exon regions) is 80%,whereas the homology between hKLK2 and hKLK1 is 65%. The deduced aminoacid sequence homology of hK2 to hK1 is 57%. Amino acid sequencesdeduced by L. J. Schedlich et al., DNA 6, 429 (1987) and B. J. Morris,Clin. Exp. Pharmacol. Physiol. 16, 345 (1989) indicate that hK2 may be atrypsin-like serine protease, whereas hK3 (PSA) is a chymotrypsin-likeserine protease. Therefore, if hK2 is indeed secretory, it may have adifferent physiological function than hK3.

The hKLK2 gene is located about 12 kbp downstream from the hKLK3 gene ina head-to-tail fashion on chromosome 19. (P. H. Riegman et al., FEBSLett., 247, 123, (1989)). The similarities of gene structure and deducedamino acid sequences of these human kallikreins suggest that theirevolution may involve the same ancestral gene. Most interestingly, asreported by Morris, cited supra; P. Chapdelaine, FEBS Lett., 236, 205(1988); and Young, Biochemistry, 31, 1952 (1992), both hK2 and hK3 maybe expressed only in the human prostate, while expression of hK1 islimited to the pancreas, submandibular gland, kidney, and othernonprostate tissues.

Tremendous interest has been generated in hK3 (PSA) because of theimportant role it plays as a marker to detect and to monitor progressionof prostate carcinoma. Its usefulness as a marker is based on theelevated serum concentration of circulating hK3 proteins which arefrequently associated with prostatic cancer. The serum concentration ofhK3 has been found to be proportional to the cancer mass in untreatedpatients, but is also proportional to the volume of hyperplastic tissuein patients with benign prostatic hyperplasia (BPH). The serum levels ofhK3 become reduced following prostate cancer therapy.

Despite the information which can be ascertained about hK2 from thegenomic DNA sequence, very little is known about the hK2 polypeptideitself. The reason for this is that the protein has not been purifiedand characterized. Thus, a need exists for a method to obtain hK2polypeptide and related polypeptides in sufficient quantity and purityfor characterization and for use as therapeutic/diagnostic agents orreagents.

SUMMARY OF THE INVENTION

The present invention provides an isolated, substantially homogenous hK2polypeptide. As used herein, in the term "hK2 polypeptide" includespre-pro hK2, pro hK2 and mature hK2 polypeptides. Pre-pro hK2 issecreted by the cell in vivo, and is cleaved during secretion to yieldpro hK2, which is then enzymatically cleaved in the extracellularenvironment to yield "mature" hK2. Most preferably, the hK2 polypeptideis contiguous in amino acid sequence with SEQ ID NO:16, SEQ ID NO:6, SEQID NO:19, or SEQ ID NO:10.

The present invention also provides isolated nucleic acid moleculesencoding hK2 polypeptide, including (a) a cDNA molecule comprising thenucleotide sequence of the coding region of the hK2 gene; (b) a DNAmolecule capable of hybridizing under stringent conditions to anucleotide sequence complementary to the nucleotide sequence of (a); and(c) a genetic variant of any of the DNA molecules of (a) and (b) whichencodes a polypeptide processing an antigenic function of naturallyoccurring hK2 polypeptide. Preferably, the nucleic acid comprises adiscrete, isolated DNA or RNA molecule encoding the complete hK2polypeptide, which can include the pre-pro, pro or mature forms. Mostpreferably, the nucleic acid is a DNA sequence contiguous with SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:20, e.g., see FIGS. 5, 6 or7. These DNA sequences can be produced using the polymerase chainreaction (PCR), and novel oligonucleotide primers employed in thesynthesis are also an embodiment of the invention.

The nucleic acid sequence also can comprise a promoter operably linkedto the nucleic acid sequence. Therefore, the invention also comprises achimeric expression vector comprising the above-described nucleic acidsequence, operationally linked to control sequences recognized by a hostcell transformed with the vector, as well as said transformed host cell,and methods of its preparation and use to produce recombinant hK2. Thus,the present invention also provides a method of using a nucleic acidmolecule, such as a cDNA clone encoding hK2 polypeptide, comprisingexpressing the nucleic acid molecule in a cultured host celltransformed, preferably stably transformed, with a chimeric expressionvector comprising said nucleic acid molecule operably linked to controlsequences recognized by the host cell transformed with the vector; andrecovering the hK2 polypeptide from the transgenic host cell, i.e., fromthe culture medium. As used herein, the term "chimeric" means that thevector comprises DNA from at least two different species, or comprisesDNA from the same species, which is linked or associated in a mannerwhich does not occur in the "native" form of said species.

More specifically, E. coli and baculovirus insect cells systems havebeen employed to produce hK2 polypeptides in two forms, i.e. pre-pro hK2(pphK2) and mature hK2 (mhK2). Thus, the present invention provides thefirst example of the overexpression of hK2 in heterologous systems.However, although pphK2 produced in E. coli has proven to be aninvaluable resource for generating antibodies to the denatured form ofthe protein, it is desirable to both discern the steps involved in thebiosynthesis of hK2 and to obtain antibodies specific for the fullyprocessed and secreted form of the protein. Therefore, mammalian cellsystems have been employed to produce hK2 polypeptides. Thus, thepresent invention also provides the first example of the expression ofhK2 in mammalian cells and purification and characterization of thesecreted protein.

The high degree of amino acid sequence homology of hK2 with hK3indicates that measuring serum concentrations of both proteins may beuseful in the diagnosis and monitoring of prostate cancer. For example,the antibodies developed against hK3 now used in these assays couldtheoretically also recognize hK2, because of mutual contamination in theantigenic preparations used to develop the anti-hK3 antibodies orbecause of antibody cross-reactivity between these two proteins. Thiscould account for the substantial percentage of false positive resultswhich are observed in current hK3 assays. On the other hand, ifcirculating hK2 levels are also elevated above baseline levels inprostate cancer patients, detection of hK2 by hK2-specific antibodiescould provide an alternative, confirmatory assay for prostate cancer.

Therefore, hK2 polypeptide, as well as variants and subunits thereof,produced by the present method can be used to produce populations ofantibodies that, in turn, can be used as the basis for assays to detectand quantify hK2 polypeptide (or "protein") in samples derived fromtissues such as prostate carcinomas, cells such as prostate cell lines,or from fluids such as seminal fluid or blood. Thus, the presentinvention also provides populations of monoclonal or polyclonalantibodies that specifically bind to hK2 polypeptide, while notsignificantly binding to hK3. The term "significantly" is defined byreference to the comparative assays discussed below. These antibodiescan also be used in affinity chromatography, to purify mammalian hK2from natural sources. The isolated, substantially homogeneous hK2 canalso be employed as a component in diagnostic assays for "native" hK2 insamples derived from human tissues or physiological fluids. For example,the recombinant hK2 can be bound to a detectable label and employed incompetitive immunoassays for hK2, as described in U.S. patentapplication Ser. No. 08/096,946, filed Jul. 22, 1993 now U.S. Pat. No.5,516,639, the disclosure of which is specifically incorporated byreference herein.

As used herein with respect to the present invention, the terms "hK2polypeptide," "hK2 protein," and "hK2" are considered to refer toidentical human materials, unless otherwise indicated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a time course study of recombinant pphK2 in sƒ9 cellsinfected with recombinant pphK2 virus. At each of the time points cellswere depleted of methionine and cysteine for 1 hour in deficient mediaand then supplemented with [³⁵ S]-methione and [³⁵ S]-cysteine. Proteinwas determined by Bradford assay. Aliquots of protein (20 μg) wereloaded onto a 12% Tris-Glycine SDS gel. A Phosphorimager cassette wasexposed overnight. The band of interest is indicated with an arrow.w.t.: wild type.

FIG. 2 depicts the detection of recombinant mhK2 in cell lysatefractions. Sƒ9 cells were infected either with recombinant mhK2, wildtype or left uninfected for 48 hours. Methionine and cysteine pools weredepleted for 1 hour in deficient media Cells were supplemented with [³⁵S]-methionine and [³⁵ S]-cysteine for 6 hours. Cells were separated intosoluble and insoluble fractions using H₂ O and repeated freeze/thawconditions. Aliquots of protein (50 μg per lane) were loaded onto a 10%Tris-Glycine SDS gel and electrophoresed. The gel was dried and exposedto x-ray film for 2 days. The band of interest is indicated with anarrow.

FIG. 3 depicts the expression of recombinant pphK2 in E. coli. E. colistrain BL21 (DE3) LysS harboring pBppHK2 was grown in LB media toO.D.₆₀₀ 0.2 and incubated without (lane 2, not-induced (N)) or with(lane 3, induced (I)) 0.4 mM IPTG for 2 hrs. Cells were lysed in samplebuffer and subjected to SDS/PAGE on a 4-20% gradient gel. Protein bandswere visualized by staining the gel with Coomassie blue.

FIG. 4 depicts the amino acid sequences of mature hK2 (deduced from cDNAsequence, SEQ ID NO:16) and hK3 (SEQ ID NO:1). Underlined sequencesdenote nonhomologous regions that can be used for preparation ofantibodies specific to hK2.

FIG. 5 depicts pphK2 cDNA containing a BamH1 site at the 5' end and aPst1 site at the 3' end (SEQ ID NO:5) (coding strand is numbered) aswell as the amino acid sequence of pre-pro hK2 encoded thereby (SEQ IDNO:6). The amino acid sequences of pro hK2 and mature hK2 are also shownon the Figure.

FIG. 6 depicts mhK2 cDNA containing an EcoR1 site at the 5' end and Pst1site at the 3' end (SEQ ID NO:7), as well as the corresponding aminoacid sequence (SEQ ID NO:8) which encompasses the amino acid sequence ofmhK2 polypeptide.

FIG. 7 depicts pro hK2 DNA (SEQ ID NO:9) (coding strand is numbered) andthe amino acid sequence of pro hK2 (SEQ ID NO:10).

FIG. 8 depicts a gel confirming the expression of recombinant pphK2 in amammalian cell line. AV12-pGThK2 (Lane 4-6) and AV12-pGT-d (Lane 3)clonal cell lines were grown in D10F media. About 300 μl of spent mediumfrom the above clones were concentrated and subjected to SDS/PAGE alongwith See Blue MW marker (lane 1) and pphK2 lysate from E. coli cells(lane 2). The gel was blotted onto nitrocellulose paper andimmunoblotted using a 1/1000 dilution of anti-pphK2 rabbit antiserum.HRP-goat anti-rabbit was used as the secondary probe and the blot wasdeveloped by DAB plus H₂ O₂. Lane 3 (AV12-pGT-d) is AV12 transfectedwith vector without insert.

FIG. 9 depicts the DEAE chromatography of AV12 media. The sample wasapplied in a bicarbonte buffer, pH 8 and eluted with a salt gradient.The solid line is the A280 elution profile. The diamond line representsthe ELISA assay of individual samples which had been dried ontomicrotiter plates and developed with rabbit anti-hK2 antibody.

FIG. 10 depicts the hydrophobic interaction profile of DEAE fractions.The fractions were pooled, concentrated and applied to an HIC column in1.2 M sodium sulfate, and eluted with a decreasing salt gradient. Thesolid line is A₂₈₀ and the diamond line shows the ELISA assay profile ofthe fractions using rabbit anti-hK2 antibody.

FIG. 11 depicts the Size Exclusion Chromatography of HIC purifiedprohK2, in particular, the A₂₈₀ profile of 22 min peak eluted off HICcolumn. The 19.4 min peak appears homogeneous by SDS-PAGE. After thispeak was lyophilized, the N-terminal sequence and amino acid compositionconfirmed its identity as the pro form of hK2.

FIG. 12 depicts the SDS/PAGE analysis of prohK2 and PSA. 1.5 μg ofpurified phK2 or PSA was boiled in sample buffer containing (R) or notcontaining (N) 1% BME. Samples were subjected to SDS/PAGE on a 4-20%gel. The protein bands were visualized by staining the gel with silver.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "hK2 polypeptide" preferably encompasses therecombinant pre-pro, pro and mature hK2 polypeptides. As proposedherein, a mature hK2 polypeptide having the amino acid sequence shown inFIG. 4 (SEQ ID NO:16), as well as "variant" polypeptides which share atleast 90% homology with SEQ ID NO:16 in the regions which aresubstantially homologous with hK3, i.e., which regions are notidentified by bars as shown in FIG. 4. Such hK2 polypeptides alsopossess antigenic function in common with the mature hK2 molecule ofFIG. 4, in that said polypeptides are also definable by antibodies whichbind specifically thereto, but which do not cross-react with hK3 (orhK1). Preferably, said antibodies react with antigenic sites or epitopesthat are also present on the mature hK2 molecule of FIG. 4. Antibodiesuseful to define common antigenic function are described in detail inSer. No. 08/096,946 now U.S. Pat. No. 5,516,639, i.e., polyclonalantisera prepared in vivo against hK2 submit 41-56.

"Isolated hK2 nucleic acid" is RNA or DNA containing greater than 15,preferably 20 or more, sequential nucleotide bases that encode abiologically active hK2 polypeptide or a variant fragment thereof, thatis complementary to the noncoding strand of the native hK2 polypeptideRNA or DNA, or hybridizes to said RNA or DNA and remains stably boundunder stringent conditions. Thus, the RNA or DNA is isolated in that itis free from at least one contaminating nucleic acid with which it isnormally associated in the natural source and is preferablysubstantially free of any other mammalian RNA or DNA. The phrase "freefrom at least one contaminating source nucleic acid with which it isnormally associated" includes the case where the nucleic acid isreintroduced into the source or natural cell but is in a differentchromosomal location or is otherwise flanked by nucleic acid sequencesnot normally found in the source cell. An example of isolated hK2nucleic acid is RNA or DNA that encodes a biologically active hK2polypeptide sharing at least 90% sequence identity with thehK3-homologous regions of the hK2 peptide of FIG. 4, as described above.The term "isolated, substantially homogenous" as used with respect to anhK2 polypeptide is defined in terms of the methodologies discussedherein below.

As used herein, the term "recombinant nucleic acid," i.e., "recombinantDNA" refers to a nucleic acid, i.e., to DNA that has been derived orisolated from any appropriate tissue source, that may be subsequentlychemically altered in vitro, an later introduced into target host cells,such as cells derived from animal, plant, insect, yeast, fungal orbacterial sources. An example of recombinant DNA "derived" from asource, would be a DNA sequence that is identified as a useful fragmentencoding hK2, or a fragment or variant thereof, and which is thenchemically synthesized in essentially pure form. An example of such DNA"isolated" from a source would be a useful DNA sequence that is excisedor removed from said source by chemical means, e.g, by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering.

Therefore, "recombinant DNA" includes completely synthetic DNAsequences, semi-synthetic DNA sequences, semi-synthetic DNA sequences,DNA sequences isolated from biological sources, and DNA sequencesderived from introduced RNA, as well as mixtures thereof. Generally, therecombinant DNA sequence is not originally resident in the genome of thehost target cell which is the recipient of the DNA, or it is resident inthe genome but is not expressed.

The recombinant DNA sequence, used for transformation herein, may becircular or linear, double-stranded or single-stranded. Generally, theDNA sequence is in the form of chimeric DNA, such as plasmid DNA, thatcan also contain coding regions flanked by control sequences whichpromote the expression of the recombinant DNA present in the resultantcell line. For example, the recombinant DNA may itself comprise apromoter that is active in mammalian cells, or may utilize a promoteralready present in the genome that is the transformation target. Suchpromoters include the CMV promoter, as well as the SV 40 late promoterand retroviral LTRs (long terminal repeat elements). Aside fromrecombinant DNA sequences that serve as transcription units for hK2 orportions thereof, a portion of the recombinant DNA may be untranscribed,serving a regulatory or a structural function.

"Control sequences" is defined to mean DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotic cells,for example, include a promoter, and optionally an operator sequence,and a ribosome binding site. Eukaryotic cells are known to utilizepromoters, polyadenylation signals, and enhancers.

"Operably linked" is defined to mean that the nucleic acids are placedin a functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, "operably linked" means that the DNA sequences being linkedare contiguous and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

Aside from recombinant DNA sequences that serve as transcription unitsfor hK2 or portions thereof, a portion of the recombinant DNA may beuntranscribed, serving a regulatory or a structural function.

The recombinant DNA to be introduced into the cells further willgenerally contain either a selectable marker gene or a reporter gene orboth to facilitate identification and selection of transformed cellsfrom the population of cells sought to be transformed. Alternatively,the selectable marker may be carried on a separate piece of DNA and usedin a co-transformation procedure. Both selectable markers and reportergenes may be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers are well knownin the art and include, for example, antibiotic and herbicide-resistancegenes, such as neo, hpt, dhfr, aroA, dapA and the like.

Reporter genes are used for identifying potentially transformed cellsand for evaluating the functionality of regulatory sequences. Reportergenes which encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene which is not present in orexpressed by the recipient organism or tissue and which encodes aprotein whose expression is manifested by some easily detectableproperty, e.g., enzymatic activity. Preferred genes include thechloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli thebeta-glucuronidase gene (gus) of the uidA locus of E. coli, and theluciferase gene from firefly Photinus pyralis. Expression of thereporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells.

Other elements functional in the host cells, such as introns, enhancers,polyadenylation sequences and the like, may also be a part of therecombinant DNA. Such elements may or may not be necessary for thefunction of the DNA, but may provide improved expression of the DNA byaffecting transcription, stability of the mRNA, or the like. Suchelements may be included in the DNA as desired to obtain the optimalperformance of the transforming DNA in the cell.

The general methods for constructing recombinant DNA which can transformtarget cells are well known to those skilled in the art, and the samecompositions and methods of construction may be utilized to produce theDNA useful herein. For example, J. Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989),provides suitable methods of construction.

The recombinant DNA can be readily introduced into the target cells bytransfection with an expression vector comprising cDNA encoding hK2, forexample, by the modified calcium phosphate precipitation procedure of C.Chen et al., Mol. Cell. Biol., 7, 2745 (1987). Transfection can also beaccomplished by lipofectin, using commercially available kits, e.g.,provided by BRL.

Suitable host cells for the expression of hK2 polypeptide are derivedfrom multicellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6; 47 (1988);Miller et al., in Genetic Engineering, J. K Setlow et al., eds., Vol. 8(Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature,315:592 (1985). A variety of viral strains for transfection are publiclyavailable, e.g., the L-1 variant of Autographa californica NPV and theBm-5 strain of Bombyx mori NPV, and such viruses may be used, preferablyfor transfection of Spodoptera frugiperda cells.

Recovery or isolation of a given fragment of DNA from a restrictiondigest can empoly separation of the digest on polyacrylamide or agarosegel by electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. For example, see Lawn etal., Nucleic Acids Res. 9, 6103-6114 (1981), and Goeddel et al., NucleicAcids Res., 8, 4057 (1980).

"Southern analysis" or "Southern blotting" is a method by which thepresence of DNA sequences in a restriction endonuclease digest of DNA orDNA-containing composition is confirmed by hybridization to a known,labeled oligonucleotide or DNA fragment. Southern analysis typicallyinvolves electrophoretic separation of DNA digests on agarose gels,denaturation of the DNA after electrophoretic separation, and transferof the DNA to nitrocellulose, nylon, or another suitable membranesupport for analysis with a radiolabeled, biotinylated, orenzyme-labeled probe as described in sections 9.37-9.52 of Sambrook etal., supra.

"Northern analysis" or "Northern blotting" is a method used to identifyRNA sequences that hybridize to a known probe such as anoligonucleotide, DNA fragment, cDNA or fragment thereof, or RNAfragment. The probe is labeled with a radioisotope such as 32-P, bybiotinylation or with an enzyme. The RNA to be analyzed can be usuallyelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe, using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,supra.

"Polymerase chain reaction" or "PCR" refers to a procedure or techniquein which amounts of a preselected piece of nucleic acid, RNA and/or DNA,are amplified as described in U.S. Pat. No. 4,683,195. Generally,sequence information from the ends of the region of interest or beyondis employed to design oligonucleotide primers. These primers will beidentical or similar in sequence to opposite strands of the template tobe amplified. PCR can be used to amplify specific RNA sequences,specific DNA sequences from total genomic DNA, and cDNA transcribed fromtotal cellular RNA, bacteriophage or plasmid sequences, and the like.See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51,263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

"Stringent conditions" are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50° C., or (2) employduring hybridization a denaturing agent such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

When hK2 polypeptide is expressed in a recombinant cell other than oneof human origin, the hK2 polypeptide is completely free of proteins orpolypeptides of human origin. However, it is necessary to purity hK2polypeptide from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogeneous as to hK2 polypeptide.For example, the culture medium or lysate can be centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions arethen separated. The hK2 polypeptide may then be purified from thesoluble protein fraction and, if necessary, from the membrane fractionof the culture lysate. HK2 polypeptide can then be purified fromcontaminant soluble proteins and polypeptides by fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; or ligand affinitychromatography.

Once isolated from the resulting transgenic host cells, derivatives andvariants of the hK2 polypeptide can be readily prepared. For example,amides of the hK2 polypeptides of the present invention may also beprepared by techniques well known in the art for converting a carboxylicacid group or precursor, to an amide. A preferred method for amideformation at the C-terminal carboxyl group is to cleave the polypeptidefrom a solid support with an appropriate amine, or to cleave in thepresence of an alcohol, yielding an ester, followed by aminolysis withthe desired amine.

Salts of carboxyl groups of the hK2 polypeptide may be prepared in theusual manner by contacting the peptide with one or more equivalents of adesired base such as, for example, a metallic hydroxide base, e.g.,sodium hydroxide; a metal carbonate or bicarbonate base such as, forexample, sodium carbonate or sodium bicarbonate; or an amine base suchas, for example, triethylamine, triethanolamine, and the like.

N-acyl derivatives of an amino group of the present polypeptides may beprepared by utilizing an N-acyl protected amino acid for the finalcondensation, or by acylating a protected or unprotected peptide. O-acylderivatives may be prepared, for example, by acylation of a free hydroxypeptide or peptide resin. Either acylation may be carried out usingstandard acylating reagents such as acyl halides, anhydrides, acylimidazoles, and the like. Both N- and O-acylation may be carried outtogether, if desired. In addition, the internal hK2 amino acid sequenceof FIG. 4 can be modified by substituting one or two conservative aminoacid substitutions for the positions specified, including substitutionswhich utilize the D rather than L form. The invention is also directedto variant or modified forms of the hK2 polypeptide of FIG. 4. One ormore of the residues of this polypeptide can be altered, so long asantigenic function is retained. Conservative amino acid substitutionsare preferred--that is, for example, aspartic-glutamic as acidic aminoacids; lysine/arginine/histidine as basic amino acids;leucine/isoleucine, methionine/valine as hydrophobic amino acids;serine/glycine/alanine/threonine as hydrophilic amino acids.

Acid addition salts of the polypeptides may be prepared by contactingthe polypeptide with one or more equivalents of the desired inorganic ororganic acid, such as, for example, hydrochloric acid. Esters ofcarboxyl groups of the polypeptides may also be prepared by any of theusual methods known in the art.

Once isolated, hK2 polypeptide and its antigenically active variants,derivatives and fragments thereof can be used in assays for hK2 insamples derived from biological materials suspected of containing hK2 oranti-hK2 antibodies, as disclosed in detail in Ser. No. 08/096,946, nowU.S. Pat. No. 5,516,639. For example, the hK2 polypeptide can belabelled with a detectable label, such as via one or more radiolabelledpeptidyl residues, and can be used to compete with endogenous hK2 forbinding to anti-hK2 antibodies, i.e., as a "capture antigen" to bind toanti-hK2 antibodies in a sample of a physiological fluid, via variouscompetitive immunoassay format for hK2 which uses immobilized anti-hK2antibodies is carried out by:

(a) providing an amount of anti-hK2 antibodies attached to a solidsurface;

(b) mixing the sample of physiological fluid to be tested with a knownamount of hK2 polypeptide which comprises a detectable label, to producea mixed sample;

(c) contacting said antibodies on said solid surface with said mixedsample for a sufficient time to allow immunological reactions to occurbetween said antibodies and said hK2, and between said antibodies andsaid labelled polypeptide;

(d) separating the solid surface from the mixed sample;

(e) detecting or determining the presence or amount of labelledpolypeptide either bound to the antibodies on the solid surface orremaining in the mixed sample; and

(f) determining from the result in step (e) the presence or amount ofsaid hK2 in said sample.

In another format which can detect endogenous hK2 in a sample by acompetitive inhibition immunoassay, a known amount of anti-hK2 antibodyis added to a sample containing an unknown amount of endogenous hK2. Theknown amount is selected to be less than the amount required to complexall of the hK2 suspected to be present, e.g., that would be present in asample of the same amount of physiological fluid obtained from a patientknown to be prostate cancer. Next, a known amount of the hK2 polypeptideof the invention or a subunit thereof, comprising a detectable label isadded. If endogenous hK2 is present in the sample, fewer antibodies willbe available to bind the labelled hK2 polypeptide, and it will remainfree in solution. If no endogenous hK2 is present, the added labelledpolypeptide will complex with the added anti-hK2 antibodies to formbinary complexes. Next, the binary antibody-antigen complexes areprecipitated by an anti-mammal IgG antibody (sheep, goat, mouse, etc.).The amount of radioactivity or other label in the precipitate (a ternarycomplex) is inversely proportional to the amount of endogenous hK2 thatis present in the sample, e.g., a pellet containing reduced amounts ofradioactivity is indicative of the presence of endogenous hK2.

Alternatively to the conventional techniques for preparing polyclonalantibodies or antisera in laboratory and farm animals, monoclonalantibodies against hK2 polypeptide can be prepared using known hybridomacell culture techniques. In general, this method involves prepared anantibody-producing fused cell line, e.g., of primary spleen cells fusedwith a compatible continuous line of myeloma cells, and growing thefused cells either in mass culture or in an animal species from whichthe myeloma cell line used was derived or is compatible. Such antibodiesoffer many advantages in comparison to those produced by inoculation ofanimals, as they are highly specific and sensitive and relatively "pure"immunochemically. Immunologically active fragments of the presentantibodies are also within the scope of the present invention, e.g., thef(ab) fragment, as are partially humanized monoclonal antibodies.

The invention will be further described by reference to the followingdetailed examples.

EXAMPLE 1 Construction of hK2 Expression Vectors

(A) Generation of recombinant baculoviruses containing pphK2 and mhK2coding sequences

A cDNA (approximately 820 bp long) encoding the entire prepro-hK2(pphK2) (from nucleotide #40 to #858 relative to the start site of thepphK2 transcript), as shown in FIG. 5, was synthesized from RNA of humanBPH tissue using reverse-transcription polymerase chain reaction(RT-PCR) technology with a pair of hK2 specific oligonucleotide primers(5'ACGCGGATCCAGCATGTGGGACCTGGTTCTCT3' SEQ ID NO:2 and5'ACAGCTGCAGTTTACTAGAGGTAGGGGTGGGAC3' SEQ ID NO:3). This cDNA wasgenerated such that 5' and 3' ends (with respect to pphK2 sensesequence) were bracketed with BamH1 and Pst 1 sequences respectively.The cDNA was then purified by agarose gel electrophoresis, and digestedwith BamH1 and Pst 1 restriction enzymes. The restricted cDNA wasligated with the BamH1-Pst 1 digested pVL1393 plasmid vector andtransformed into the E. coli HB101 strain. E. coli harboring pphK2cDNA/pVL1393 plasmid vector were selected and verified by restrictionenzyme mapping and DNA sequencing. Plasmid pphK2 cDNA/pVL1393 wasmass-produced in E. coli and purified by CsCl gradientultra-centrifugation.

cDNA encoding the mature hK2 was synthesized using PCR with theaforementioned pphK2 cDNA as the template plus a pair of hK2oligonucleotides (5'ACGCGGATCCAGCATGTGGGACCTGGTTCTCT3' SEQ ID NO:2 and5'ACCGGAATTCATGATTGTGGGAGGCTGGGAGTGT3' SEQ ID NO:4). As noted, the 3'end oligonucleotide was the same 3' end oligonucleotide used forsynthesizing the pphK2 cDNA. However, the 5' end oligonucleotide wasdifferent from the 5' oligonucleotide used for the pphK2 cDNA, andtherefore generates a cDNA coding for the mature form of hK2 (mhK2), asshown in FIG. 6. The mhK2 cDNA was bracketed with EcoR1 and Pst1sequences at the 5' and 3' ends respectively. The protein produced fromthe mhK2 cDNA will gain an exogenous methionine at its N-terminus. ThemhK2/pVL1393 vector was generated and purified as described forpphK2/pVL1393. The DNA sequence analysis for pphK2 and mhK2 in pVL1393showed that one nucleotide (#805) has been altered (G to T) in a silentmutation.

pphK2/pVL1393 or mhK2/pVL1393 DNA (2 μg) were cotransfected with alinearized Baculogold DNA (0.5 μg; Pharmingen, San Diego, Calif.) intoSƒ9 insect cells according to Pharmingen instructions (S. Gruenwold etal., baculovirus expression vector system Procedures and Methods Manual,Pharmingen, San Diego, Calif. (1993)). Four to six days after thetransfection, Sƒ9 cell spent medium containing viral particles washarvested and used to infect fresh Sƒ9 cells to amplify viral titers.Total RNA was isolated for Northern blot analysis of authentic pphK2 ormhK2 transcript using hK2 cDNA as a probe. Further proof of pphK2 ormhK2 transcript expressed in recombinant virus infected Sƒ9 cells wasobtained by RT-PCR and DNA sequencing. Pure recombinant baculoviruscontaining pphK2 or mhK2 were obtained by secondary or tertiary plaquepurification protocol according to instructions from Pharmingen (S.Gruenwold et al., cited above).

EXAMPLE 2 Generation of Prokaryotic Expression Vector

A 0.8 kb fragment representing the entire preprohK2 (pphK2) codingsequence was generated by polymerase chain reaction (PCR) using primersA (5'TATACATATGTGGGACCTGGTTCTCTCC3' SEQ ID NO.:11) and B(5'ATATGGATCCTCAGGGGTTGGCTGCGATGGT3' SEQ ID NO:12) and plasmid pVL1393containing pphK2 as the template. The pphK2 bacterial expression vector(pBPPHK2) was prepared by standard DNA cloning technology, (Sambrook,cited above), to subclone this 0.8 kb fragment into the Nde1/BamH1 siteof the plasmid pPHS579 (a gift from Dr. H. Hsiung, Eli Lilly Co, Inc.)under the control of T7 promoter. The DNA of the entire insert plus thecloning sites was sequenced to confirm that no cloning artifacts hadoccurred and to ensure that no anomalies in the sequence had beengenerated by PCR. pBPPHK2 was transformed into E. coli BL21 (DE3)Lys S(Novagen, Inc., Madison, Wis.).

EXAMPLE 3 Generation of a Mammalian Expression Vector

To express hK2 in mammalian cell lines, a 0.8 kb fragment representingthe entire preprohk2 (pphK2) coding sequence was generated by PCR usingprimers A(5'ATATGGATCCATATGTCAGCATGTGGGACCTGGTCTCTCCA3') (SEQ ID NO:17)and B(5'ATATGGATCCTCAGGGGTTGGCTGCGATGGT3') (SEQ ID NO:12) and plasmidpVL1393 containing pphK2 as the template. The mammalian expressionvector (pGThK2) was prepared using standard DNA cloning technology(Sambrook, 1989), to clone this 0.8 kb fragment into the Bc11 site ofthe plasmid pGT-d (a gift from Dr. Brian Grinnell, Eli Lilly, Inc.)under control of the GBMT promotor. The DNA of the entire insert plusthe cloning sites was sequenced and a single base change at position 650(T for C) was noted. This change results in an amino acid substitutionof valine for alanine at position 217 in hK2. AV12-664 (ATCC CRL-9595),a cell line derived from a adenovirus-induced tumors in Syrian hamster,was grown in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum (D10F) and transfected with plasmid pGThK2 using thecalcium phosphate method.

EXAMPLE 4 Identification of Recombinant pphK2 and mhK2

A. baculovirus--insect cell system

Sƒ9 cells (7×10⁶ /plate) were seeded onto 100 mm Corning plates with 10%fetal calf serum--Graces medium at room temperature for 1 hr. Afterattachment on culture plates, cells were infected with wild type orrecombinant baculovirus in serum free Excell-400 medium and incubated at27° C. Control cells were grown in the absence of virus. At designatedtimes (24-96 hr) cells were placed in fresh Sƒ9-IIOO media deficient ofeither methionine or methionine and cysteine for 45-60 min at 27° C.,then incubated with Promix (0.143 mCi/plate; a mixture of [³⁵S]-methionine and [³⁵ S]-cysteine; 1,000-1,400 Ci/mmol; Amersham) inserum free and methionine/cysteine deficient Sƒ9IIOO medium (Biofluids)for 5-8 hr or 20 hr. After the end of each incubation time, cells andspent media were separated by centrifugation (1,000 rpm; Beckman J-6B;Beckman, Fullerton, Calif.). Cells were washed and centrifuged (13,000rpm; Biofuge 13, Baxter) twice. The washed cells were lysed byfreeze/thaw in a detergent buffer (10 mM Tris, pH 7.5; 130 mM NaCl, 1%Triton X-100, 10 mM NaF; 10 mM NaPi, 10 mM Nappi, pH7.5) or H₂ O andcentrifuged to obtain cytosol and insoluble cellular fractions. Proteincontents of the above samples were determined by either the Bradford orLowry method (BioRad, Inc., Melville, N.Y.). The above spent media,cystosol and insoluble cellular fraction were frozen and storedseparately until used. A duplicate set of samples were prepared without³⁵ S-labeling.

For SDS-polyacrylamide gel electrophoresis (PAGE) analysis of expressionof hK2 protein in Sƒ9 cells, samples were added to sample buffer (U. KLaemmli, Nature, 227, 680 (1970)), heated at 95° C. for 5 minutes andsubjected to SDS-PAGE under reducing conditions.

Northern blot analysis was routinely used to screen and isolate clonalrecombinant baculoviruses expressing pphK2 or mhK2 mRNA. A comparison ofthe corresponding lanes in both autoradiographs of the Northern blot andphotographs of ethidium bromide staining of RNA shows that mRNA forpphK2 or mhK2 was present in recombinant virus infected Sƒ9 but not inwild type virus-infected cells. Moreover, each of the pphK2 or mhK2 mRNApositive lanes represents RNA isolated from Sƒ9 cells infected withrecombinant viruses derived from a single viral plaque. Thus, theresults suggest that high frequency (100%) of recombinant baculoviruscontaining either pphK2 or mhK2 was obtained from the abovecotransfection. Furthermore, the sequences of pphK2 or mhK2 expressed inviral infected Sƒ9 cells were confirmed by a combination of RT-PCR,cloning and DNA sequencing.

To determine whether the pphK2 protein is expressed in the insect cellSƒ9, time course studies using ³⁵ S-labeling of de novo synthesis ofprotein was performed and detected by SDS denaturing polyacrylamide gelelectrophoresis (PAGE). As seen in the autoradiograph (FIG. 1), a uniqueprotein (about 28 KDa) was found in pphK2-recombinant virus-infected Sƒ9cells at 35-74 hour post-infection. This band was missing in uninfectedcells or cells infected with wild type virus. The viral polyhedronprotein (about 32 KDa) was found (FIG. 1) as expected in Sƒ9 cellsinfected with wild type virus, whereas it was not expressed byrecombinant virus (FIG. 1). The protein was detected in cytosol whensubcellular fractions (cytosol vs. insoluble fraction) was prepared bylysing cells with H₂ O and freeze-thaw, whereas this 28 KD protein wasdetected in insoluble fraction when prepared by a detergent buffer andfreeze-thaw (data not shown).

The mhK2 protein was also expressed in the insect cell Sƒ9, ³⁵S-labeling of de novo synthesized protein was performed. As seen in theautoradiograph (FIG. 2), a unique protein (about 28 KDa) was found inthe insoluble fraction of mhK2-recombinant virus-infected Sƒ9 cells at48 hours post-infection. This band was missing in uninfected cells orcells infected with wild type virus. The viral polyhedron protein (about32 KDa) was found in wild type virus-infected cells, whereas it was notexpressed in cells infected with recombinant virus (FIG. 1). When thecytosol fraction was examined, no 28 KDa band was observed.

B. E. coli system

Plasmid pBPPHK2 was transformed into E. coli BL21 (DE3) pLysS (Novagen,Inc., Madison, Wis.). This strain contains a chromosomal copy of T7 RNApolymerase under the control of inducible LacUV5 promoter. Upon additionof IPTG (isopropyl-β-D-thiogalactopyranoside) the expression of the T7RNA polymerase is induced which in turn activates the T7 promoterresulting in overproduction of the gene product under control of thispromoter. To determine whether the product of the ppHK2 gene would beexpressed from pBPPHK2, single colonies of BL21 E. coli transformed withpBPPHK2 were grown to O.D.₆₀₀ =0.2 in 10 ml LB media plus ampicillin(100 μg/ml) and induced with 0.4 mM IPTG (Sigma, Inc.). Cells wereharvested 2 hours after induction by centrifugation and resuspended in1.5 ml SDS/PAGE sample buffer (U. K. Laemmli, Nature, 227, 680 1970)before SDS/PAGE analysis. The cell pellet from the IPTG-induced culturewas resuspended in 0.05M Tris, pH 8.0 (at 9 ml/gm cell pellet) andstirred at room temperature (25° C., r.t.) for 1 hour. Lysozyme (4mg/ml) was added to this suspension (at 1 ml/gm cell pellet) and thesuspension was stirred at r.t. for 30 min followed by incubation on icefor 30 min. The suspension was sonicated for 2 min at 150 watts andcentrifuged at 3000×g to isolate the inclusion bodies. Inclusion bodieswere resuspended in running buffer (25 mM Tris, 192 mM glycine, 0.1%SDS) and after centrifugation both the pellet and the supernatant wereanalyzed by SDS/PAGE.

About 90% of the pphK2 was found to be in the supernatant fraction whichindicated that pphK2 is soluble in 0.1% SDS. To prepare samples foramino acid sequence analysis, 20 μl of inclusion body lysate wassubjected to SDS/PAGE on a 4-20% gradient gel (BIO-RAD, Inc., Melville,N.Y.). The protein was blotted from the gel onto 0.2μ PVDF paper(BIO-RAD) and stained with Coomassie blue. The protein band of interestwas cut out from the blot and subjected to amino acid sequencing using aprotein sequencer model 477A (Applied Biosystem, Inc., Foster City,Calif.).

The induced cells overproduced large amounts of a polypeptide withapparent molecular mass of about 28 kd (FIG. 3). Densitometric analysisindicated that this protein comprised approximately 40% of totalcellular protein. The size of this protein as determined by an SDS-PAGEgel was comparable to that predicted from coding sequence for pphK2. Toconfirm that this protein is pphK2, the sequence of the first 10 aminoacids (MWDLVLSIAL) (SEQ ID NO:13) from the N-terminus was determined.This sequence agrees perfectly with that deduced from the DNA sequenceof pphK2 cDNA. As noted, it has different identity from the first 10amino acids of both pphK1 (MWFLVLCLAL) (SEQ ID NO:14) and pphK3(MWVPVVFLTL) (SEQ ID NO:15). It also shows that this protein is notmodified or processed at the N-terminus either during or afterexpression in E. coli. These results demonstrate that we were able toaccurately express pphK2 in E. coli from pBPPHK2.

C. Mammalian System

1. Isolation and Purification of protein

Plasmid pGThK2 was transformed into hamster cell line AV12-664(ATCC-CRL-9595). To determine whether the product of the ppHK2 genewould be expressed from pGThK2, AV12-pGThK2 #2 was grown in D10F+200 nMMTX. At about 60% confluency the cells were washed with Hank's balancedsalt solution and resuspended in serum-free HH4 medium. The spent mediumwas collected after 7 days (serum-free spent medium) and stored at -20°C. FIG. 8 depicts a SDS-PAGE confirming expression of recombinant pphK2in a mammalian cell line. AV12-pGThK2 (Lane 4-6) and AV12-pGT-d (Lane 3)clonal cell lines were grown in D10F media About 300 μl of spent mediumfrom the above clones were concentrated and subjected to SDS/PAGE alongwith See Blue MW marker (lane 1) and pphK2 lysate from E. coli cells(lane 2). The gel was blotted onto nitrocellulose paper andimmunoblotted using a 1/1000 dilution of anti-pphK2 rabbit antiserum.HRP-goat anti-rabbit was used as the secondary probe and the blot wasdeveloped by DAB plus H₂ O₂. Lane 3 (AV12-pGT-d) is AV12 transfectedwith vector without insert.

To purify the protein, the serum-free spent medium was concentrated from5-10 fold by ultrafiltration with a 10 kDa molecular weight cutoffmembrane then dialyzed overnight at 4° C. versus 50 mM sodiumbicarbonate, pH 8. Samples were filtered with 0.2μ filters and thenpumped directly onto a TSK DEAE-5PW HPLC column (21 mm×150 mm) at a flowrate of 5 mL/min. Buffer A contained 50 mM sodium bicarbonate, pH 7.9Buffer B contained 50 mM sodium bicarbonate plus 0.5 M sodium chloride,pH 7.6. The elution profile shown in FIG. 9 was developed with agradient from 0-50% Buffer B over 35 min; 50-100% B from 35-40 min andisocratic elution at 100% B for 5 min before re-equilibration in BufferA. The flow rate was 5 mL/min throughout.

DEAE fractions were assayed for the presence of hK2 by ELISA usingrabbit anit-pphK2 as primary antibodies. The ELISA assayed showed a peakof hK2 activity which eluted at approximately 0-2M NaCl (shown as thetriangle line in FIG. 9), which correlated well with the appearance of a34 kDa band of protein seen by SDS-PAGE in the same fractions (data notshown).

Fractions with hK2 activity were pooled and concentrated byultrafiltration with 10 kDa membranes to approximately 5-8 mL where uponsolid ammonium sulfate was added to make a final concentration of 1 M.This sample was then injected onto a PolyLC. polypropyl aspartamidecolumn, 1000A pore size, 4.6 mm×200 mm, to resolve protein byhydrophobic interaction chromatography (HIC, see FIG. 10). Buffer A was20 mM Na phosphate, 1.2 M Na sulfate pH 6.3 and Buffer B was 50 mM Naphosphate, 5% 2-propanol, pH 7.4. The elution gradient was 0-20% B over5 min; 20-55% B from 5-20 min, isocratic at 55% B from 20-23 min,55-100% B from 23-25 min; isocratic at 100% B for 2 min beforere-equilibration Buffer A. The flow rate was 0.7 mL/min. Greater than90% of the A₂₈₀ was not retained on HIC column. The main peak retainedon HIC, which eluted at 22 min, also showed the highest peak of activityby ELISA assay (triangle line, FIG. 10).

HIC fractions which tested positive for hK2 on ELISA were pooled,ultrafilter concentrated as above to a volume less than 1 mL theninjected on a 10/30 Pharmacia S12 size exclusion column equilibrated in100 mM ammonium acetate. The flow rate was 0.7 mL/min. When the 22 minpeak from HIC was resolved by size exclusion chromatography, typicallyabout 80-90% of the protein A₂₈₀ eluted at 19.4 min, a retention timeconsistent with a protein of approximately 34 kDa (FIG. 11). The onlyother protein peak on SEC, eluting at 16.7 min, corresponded to an about70 KDa protein seen also in previous purification steps.

To examine the efficiency of our purification scheme, 1.5 μg of purifiedphK2 was subjected to SDS/PAGE in the presence or absence ofβ-mercaptoethanol (BME), and the gel was stained with silver. Resultsshowed that the phK2 in our sample was about 95% pure (FIG. 12). It alsoshowed that pro-hK2 migrated at about 30 KD in the absence of BME, andit migrated at about 34 kDa in the presence of BME. This pattern issimilar to that observed for the PSA purified from seminal fluid (FIG.12).

Recombinant phK2 is recognized by rabbit anti-pphK2, rabbit anti-PSA anda murine monoclonal antibody directed against a polypeptide coveringamino acids 41-56 of hK2, when analyzed on WESTERN blots or when drieddown on microtiter plates. However, phK2 was not detectable by theseantibodies in sandwich assays. These results further demonstrate thatthe phK2 and PSA are conformationally different and the antibodiescurrently available to PSA or hK2 can not detect phK2 in its nativeform. Furthermore, phK2 was not detectable by the Tandem R or free-PSAassays (immunological assays for detection of PSA in serum).

A sample of the hybridoma (HKLA 523.5) secreting the murine monoclonalantibody has been deposited in the American Type Culture Collection,Rockville, Md., and assigned ATCC HB-11876.

2. Amino Acid Analysis and Protein Sequencing of phK2

The peak collected off size exclusion chromatography (SEC) in ammoniumacetate was lyophilized to remove the buffer then reconstituted inwater. An aliquot (2.5 μg) of this sample was loaded on a Portonmembrane (Beckman instruments) and subjected to automated N-terminalsequence analysis on an Applied Biosystems model 477A protein sequencerwhich yielded the following sequence:Val-Pro-Leu-Ile-Gln-Ser-Arg-Ile-Val-Gly-Gly-Trp-Glu- (SEQ ID NO:18). Analiquot of the same sample in water was also hydrolyzed in gaseous 6 NHCI under vacuum for 20 h at 112° C. then reconstituted in 0.1N HCI andanalyzed on an Hewlett Packard Aminoquant amino acid analyzer utilizingpre-column derivatization of amino acids with OPA for primary and FMOCfor secondary amines.

No competing sequence was evident from the profile of amino acidsreleased sequentially by the Edman degradation procedure. By analogy toPSA this protein is pro hK2, since the known sequence of mature PSA hasbeen shown to begin with Ileu-Val-Gly-etc and pro PSA has beenpostulated to have an extra 7 amino acids at the N-terminus. Amino acidanalysis of this protein yielded an amino acid composition consistentwith the recombinant sequence of prohK2. These results demonstrate thatpphK2 having SEQ ID NO:19 was accurately expressed in the mammalian cellline AV12-664 from pGThK2.

EXAMPLE 5 Production of antibodies to recombinant pphK2

A. E. coli System

To prepare pphK2 for rabbit immunization, the inclusion bodies obtainedfrom bacterial cultures of BL21 (pBpphK2) after IPTG induction as inExample 4B were resuspended in 100 μl SDS/PAGE sample buffer/mlbacterial culture and electrophoresed on preparative SDS/PAGE. The pphK2band was excised and electroeluted from the gel into running buffer (25mM Tris, 192 mM glycine, 0.1% SDS) and used as the immunogen. Tworabbits were each immunized with 100 μg of the immunogen in completeFreund's adjuvant and were boosted twice in three week intervals with100 μg of the immunogen in incomplete Freund's adjuvant and PBS,respectively. Rabbit anti-pphK2 sera was obtained one week following thesecond boost. The presence of anti-pphK2 in the rabbit antiserum wasshown by ELISA (data not shown). Once confirmed by this method, thehighest titer antiserum was tested on Western blots using lysates fromIPTG induced or non-induced cultures of BI.21 (pBpphK2). It was evidentthat the antiserum contained antibodies highly specific for the pphK2protein since a protein band at about 28 kd corresponding to pphK2 wasrecognized only in the induced lysate. The antiserum also recognized thepurified pphK2 further showing the specificity of the antibodies topphK2. The above data demonstrate that the antibodies recognize theprepro-form of hK2.

To delineate if the antiserum recognizes the mature form of hK2 (mhK2),mhK2 was expressed in E. coli as a glutathione S-transferase fusionprotein (GST-mhK2, 58 kd), and the cell lysate was immunoblotted usinganit-pphK2 rabbit antiserum. It was evident that anti-pphK2 antiserumrecognized the GST-mkK2, demonstrating that antibodies were at least inpart against the mature region of pphK2. To examine the patternrecognized in seminal fluid by anti-pphK2 antibodies, seminal fluid wasprepared from pooled semen as described by Sensabaugh and Blake, J.Urolog 149, 1523 (1990), and immunoblotted with anti-pphK2 rabbitantiserum. The antiserum recognized a major band at about 34 kd plusseveral minor bands at lower MW. The pre-immune serum did not recognizeany bands in any of the above experiments, showing that the antibodieswere generated by immunization.

To determine whether there are any pphK2-specific antibodies in rabbitanit-pphK2 antiserum, the antibodies cross-reacting to PSA were absorbedout of the antiserum by a PSA affinity resin. Specifically, 1 ml of thesera was diluted with 1 mL 100 mM HEPES, pH. 7.5 and incubated withnative PSA-bound Affigel-10 for 3.5 hours at 4° C. The mixture was usedpoured into a column, the flow-through was collected and the column waswashed with 30 ml HEPES buffer. Antibodies bound to the column (eluate)were eluted by acetic acid (1N, pH 4.0) and neutralized to pH. 6.6 withNH₄ OH. Native PSA was isolated from seminal fluid as described bySensabaugh and Blake, cited above. ppPSA was purified from E. colitransformed with plasmid pPHS579 (containing ppPSA under control of T7promoter) using a procedure analogous to pphK2 purification.

The flow-through and the column eluate were tested for Abs recognizingpphK2, ppPSA and native PSA (PSA isolated from seminal fluid) usingWestern blot analysis. It was evident that antibodies in the untreatedrabbit anti-pphK2 antiserum recognized all three proteins indicatingthat pphK2, ppPSA and seminal fluid-PSA share some similar epitopes.However, while the column eluate contained antibodies that recognizedall three protein, the flow-through contained antibodies that recognizedonly pphK2. This indicates that anti-pphK2 antiserum containspphK2-specific antibodies and these antibodies can be isolated by PSAaffinity absorption. This system enabled us to generate anti-pphK2antibodies which recognize both pphK2 and mhK2. Thus, utilizingimmunogenic and pure recombinant hK2 protein, generate rabbit antiserumwas generated which contains pphK2-specific antibodies, providing avaluable source for generating and screening for hK2-specific monoclonalantibodies.

These examples describe the use of three heterologous expression systems(i.e. both prokaryotic and eukaryotic) for the successful expression ofthe hK2 polypeptide. Thus, the method of the invention enablesproduction of large quantities of substantially pure hK2 polypeptide.The polypeptide can be used both to study its biological functions andto produce immunodetection reagents such as labelled hK2 polypeptide,labelled fragments thereof and antibodies thereto. The immunoreagentscan provide a method to purify native hK2 and to study the properties ofthe purified native hK2 polypeptide.

The pphK2 overproduced in E. coli can be readily solubilized in 0.1%SDS, thus solubility is not a problem. This is in contrast to theexpression of human salivary kallikrein protein, hK1, in E. coli, whichwas found in insoluble inclusion bodies (J. Wang, et al Biochem. J.,276, 63 (1991)). In contrast, the present invention yields almost pureprotein which can be purified to homogeneity by preparative SDS-PAGE.This purified recombinant pphK2 can be used for the generation ofmonoclonal and polyclonal antibodies.

As shown above, Baculogold viral DNA can be used to generate arecombinant baculovirus containing pphK2 or mhK2. Use of Baculogoldviral DNA provides high selection of positive recombinant baculoviruses.Indeed, Northern blot analysis showed a high frequency of recombinantvirus expressing pphK2 or mhK2 mRNA. Moreover, SDS-PAGE analysis showedthat both pphK2 and mhK2 recombinant viruses produced unique proteinswith sizes similar to the calculated molecular weights for pphK2 ormhK2. Although the levels of the recombinant hK2 expressed in insectsystem may not be as high as in E. coli, the hK2 protein produced inbaculovirus-insect system may contain the secreted form which would bemore like the natural form of the protein.

Plasmids pphK2/pVL1393 in E. col. H13101 has been deposited in theAmerican Type Culture Collection, Rockville, Md., USA on May 2, 1994under the provisions of the Budapest Treaty and have been assignedaccession number ATCC 69614.

All publications, patents and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 21                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 237 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Ile Val Gly Gly Trp Glu Cys Glu Lys His Se - #r Gln Pro Trp Gln        Val                                                                             1               5   - #                10  - #                15              - - Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gl - #y Gly Val Leu Val His                  20      - #            25      - #            30                   - - Pro Gln Trp Val Leu Thr Ala Ala His Cys Il - #e Arg Asn Lys Ser Val              35          - #        40          - #        45                       - - Ile Leu Leu Gly Arg His Ser Leu Phe His Pr - #o Glu Asp Thr Gly Gln          50              - #    55              - #    60                           - - Val Phe Gln Val Ser His Ser Phe Pro His Pr - #o Leu Tyr Asp Met Ser      65                  - #70                  - #75                  - #80        - - Leu Leu Lys Asn Arg Phe Leu Arg Pro Gly As - #p Asp Ser Ser His Asp                      85  - #                90  - #                95               - - Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Gl - #u Leu Thr Asp Ala Val                  100      - #           105      - #           110                  - - Lys Val Met Asp Leu Pro Thr Gln Glu Pro Al - #a Leu Gly Thr Thr Cys              115          - #       120          - #       125                      - - Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Gl - #u Glu Phe Leu Thr Pro          130              - #   135              - #   140                          - - Lys Lys Leu Gln Cys Val Asp Leu His Val Il - #e Ser Asn Asp Val Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Gln Val His Pro Gln Lys Val Thr Lys Ph - #e Met Leu Cys Ala        Gly                                                                                             165  - #               170  - #               175             - - Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser Gl - #y Asp Ser Gly Gly Pro                  180      - #           185      - #           190                  - - Leu Val Cys Asn Gly Val Leu Gln Gly Ile Th - #r Ser Trp Gly Ser Glu              195          - #       200          - #       205                      - - Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Ty - #r Thr Lys Val Val His          210              - #   215              - #   220                          - - Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Al - #a Asn Pro                  225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - ACGCGGATCC AGCATGTGGG ACCTGGTTCT CT       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - ACAGCTGCAG TTTACTAGAG GTAGGGGTGG GAC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - ACCGGAATTC ATGATTGTGG GAGGCTGGGA GTGT       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 832 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 10..792                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GGATCCAGC ATG TGG GAC CTG GTT CTC TCC ATC GCC - # TTG TCT GTG GGG             48                                                                                Met Trp Asp Leu Val - #Leu Ser Ile Ala Leu Ser Val Gly                          1      - #         5         - #         10                        - - TGC ACT GGT GCC GTG CCC CTC ATC CAG TCT CG - #G ATT GTG GGA GGC TGG           96                                                                       Cys Thr Gly Ala Val Pro Leu Ile Gln Ser Ar - #g Ile Val Gly Gly Trp                15             - #     20             - #     25                          - - GAG TGT GAG AAG CAT TCC CAA CCC TGG CAG GT - #G GCT GTG TAC AGT CAT          144                                                                       Glu Cys Glu Lys His Ser Gln Pro Trp Gln Va - #l Ala Val Tyr Ser His            30                 - # 35                 - # 40                 - # 45       - - GGA TGG GCA CAC TGT GGG GGT GTC CTG GTG CA - #C CCC CAG TGG GTG CTC          192                                                                       Gly Trp Ala His Cys Gly Gly Val Leu Val Hi - #s Pro Gln Trp Val Leu                            50 - #                 55 - #                 60              - - ACA GCT GCC CAT TGC CTA AAG AAG AAT AGC CA - #G GTC TGG CTG GGT CGG          240                                                                       Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gl - #n Val Trp Leu Gly Arg                        65     - #             70     - #             75                  - - CAC AAC CTG TTT GAG CCT GAA GAC ACA GGC CA - #G AGG GTC CCT GTC AGC          288                                                                       His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gl - #n Arg Val Pro Val Ser                    80         - #         85         - #         90                      - - CAC AGC TTC CCA CAC CCG CTC TAC AAT ATG AG - #C CTT CTG AAG CAT CAA          336                                                                       His Ser Phe Pro His Pro Leu Tyr Asn Met Se - #r Leu Leu Lys His Gln                95             - #    100             - #    105                          - - AGC CTT AGA CCA GAT GAA GAC TCC AGC CAT GA - #C CTC ATG CTG CTC CGC          384                                                                       Ser Leu Arg Pro Asp Glu Asp Ser Ser His As - #p Leu Met Leu Leu Arg           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - CTG TCA GAG CCT GCC AAG ATC ACA GAT GTT GT - #G AAG GTC CTG GGC        CTG      432                                                                    Leu Ser Glu Pro Ala Lys Ile Thr Asp Val Va - #l Lys Val Leu Gly Leu                          130  - #               135  - #               140              - - CCC ACC CAG GAG CCA GCA CTG GGG ACC ACC TG - #C TAC GCC TCA GGC TGG          480                                                                       Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cy - #s Tyr Ala Ser Gly Trp                       145      - #           150      - #           155                  - - GGC AGC ATC GAA CCA GAG GAG TTC TTG CGC CC - #C AGG AGT CTT CAG TGT          528                                                                       Gly Ser Ile Glu Pro Glu Glu Phe Leu Arg Pr - #o Arg Ser Leu Gln Cys                   160          - #       165          - #       170                      - - GTG AGC CTC CAT CTC CTG TCC AAT GAC ATG TG - #T GCT AGA GCT TAC TCT          576                                                                       Val Ser Leu His Leu Leu Ser Asn Asp Met Cy - #s Ala Arg Ala Tyr Ser               175              - #   180              - #   185                          - - GAG AAG GTG ACA GAG TTC ATG TTG TGT GCT GG - #G CTC TGG ACA GGT GGT          624                                                                       Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gl - #y Leu Trp Thr Gly Gly           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - AAA GAC ACT TGT GGG GGT GAT TCT GGG GGT CC - #A CTT GTC TGT AAT        GGT      672                                                                    Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pr - #o Leu Val Cys Asn Gly                          210  - #               215  - #               220              - - GTG CTT CAA GGT ATC ACA TCA TGG GGC CCT GA - #G CCA TGT GCC CTG CCT          720                                                                       Val Leu Gln Gly Ile Thr Ser Trp Gly Pro Gl - #u Pro Cys Ala Leu Pro                       225      - #           230      - #           235                  - - GAA AAG CCT GCT GTG TAC ACC AAG GTG GTG CA - #T TAC CGG AAG TGG ATC          768                                                                       Glu Lys Pro Ala Val Tyr Thr Lys Val Val Hi - #s Tyr Arg Lys Trp Ile                   240          - #       245          - #       250                      - - AAG GAC ACC ATC GCA GCC AAC CCC TGAGTGCCCC TG - #TCCCACCC CTACCTCTAG         822                                                                       Lys Asp Thr Ile Ala Ala Asn Pro                                                   255              - #   260                                                 - - TAAACTGCAG                - #                  - #                      - #       832                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 261 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Trp Asp Leu Val Leu Ser Ile Ala Leu Se - #r Val Gly Cys Thr Gly        1               5 - #                 10 - #                 15              - - Ala Val Pro Leu Ile Gln Ser Arg Ile Val Gl - #y Gly Trp Glu Cys Glu                   20     - #             25     - #             30                  - - Lys His Ser Gln Pro Trp Gln Val Ala Val Ty - #r Ser His Gly Trp Ala               35         - #         40         - #         45                      - - His Cys Gly Gly Val Leu Val His Pro Gln Tr - #p Val Leu Thr Ala Ala           50             - #     55             - #     60                          - - His Cys Leu Lys Lys Asn Ser Gln Val Trp Le - #u Gly Arg His Asn Leu       65                 - # 70                 - # 75                 - # 80       - - Phe Glu Pro Glu Asp Thr Gly Gln Arg Val Pr - #o Val Ser His Ser Phe                       85 - #                 90 - #                 95              - - Pro His Pro Leu Tyr Asn Met Ser Leu Leu Ly - #s His Gln Ser Leu Arg                  100      - #           105      - #           110                  - - Pro Asp Glu Asp Ser Ser His Asp Leu Met Le - #u Leu Arg Leu Ser Glu              115          - #       120          - #       125                      - - Pro Ala Lys Ile Thr Asp Val Val Lys Val Le - #u Gly Leu Pro Thr Gln          130              - #   135              - #   140                          - - Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Se - #r Gly Trp Gly Ser Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Le - #u Gln Cys Val Ser        Leu                                                                                             165  - #               170  - #               175             - - His Leu Leu Ser Asn Asp Met Cys Ala Arg Al - #a Tyr Ser Glu Lys Val                  180      - #           185      - #           190                  - - Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Th - #r Gly Gly Lys Asp Thr              195          - #       200          - #       205                      - - Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cy - #s Asn Gly Val Leu Gln          210              - #   215              - #   220                          - - Gly Ile Thr Ser Trp Gly Pro Glu Pro Cys Al - #a Leu Pro Glu Lys Pro      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Val Tyr Thr Lys Val Val His Tyr Arg Ly - #s Trp Ile Lys Asp        Thr                                                                                             245  - #               250  - #               255             - - Ile Ala Ala Asn Pro                                                                  260                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 760 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..720                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GAATTC ATG ATT GTG GGA GGC TGG GAG TGT GAG - #AAG CAT TCC CAA CCC            48                                                                               Met Ile Val Gly Gly Trp Gl - #u Cys Glu Lys His Ser Gln Pro                     1         - #      5            - #      10                           - - TGG CAG GTG GCT GTG TAC AGT CAT GGA TGG GC - #A CAC TGT GGG GGT GTC           96                                                                       Trp Gln Val Ala Val Tyr Ser His Gly Trp Al - #a His Cys Gly Gly Val            15                 - # 20                 - # 25                 - # 30       - - CTG GTG CAC CCC CAG TGG GTG CTC ACA GCT GC - #C CAT TGC CTA AAG AAG          144                                                                       Leu Val His Pro Gln Trp Val Leu Thr Ala Al - #a His Cys Leu Lys Lys                            35 - #                 40 - #                 45              - - AAT AGC CAG GTC TGG CTG GGT CGG CAC AAC CT - #G TTT GAG CCT GAA GAC          192                                                                       Asn Ser Gln Val Trp Leu Gly Arg His Asn Le - #u Phe Glu Pro Glu Asp                        50     - #             55     - #             60                  - - ACA GGC CAG AGG GTC CCT GTC AGC CAC AGC TT - #C CCA CAC CCG CTC TAC          240                                                                       Thr Gly Gln Arg Val Pro Val Ser His Ser Ph - #e Pro His Pro Leu Tyr                    65         - #         70         - #         75                      - - AAT ATG AGC CTT CTG AAG CAT CAA AGC CTT AG - #A CCA GAT GAA GAC TCC          288                                                                       Asn Met Ser Leu Leu Lys His Gln Ser Leu Ar - #g Pro Asp Glu Asp Ser                80             - #     85             - #     90                          - - AGC CAT GAC CTC ATG CTG CTC CGC CTG TCA GA - #G CCT GCC AAG ATC ACA          336                                                                       Ser His Asp Leu Met Leu Leu Arg Leu Ser Gl - #u Pro Ala Lys Ile Thr            95                 - #100                 - #105                 - #110       - - GAT GTT GTG AAG GTC CTG GGC CTG CCC ACC CA - #G GAG CCA GCA CTG GGG          384                                                                       Asp Val Val Lys Val Leu Gly Leu Pro Thr Gl - #n Glu Pro Ala Leu Gly                           115  - #               120  - #               125              - - ACC ACC TGC TAC GCC TCA GGC TGG GGC AGC AT - #C GAA CCA GAG GAG TTC          432                                                                       Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Il - #e Glu Pro Glu Glu Phe                       130      - #           135      - #           140                  - - TTG CGC CCC AGG AGT CTT CAG TGT GTG AGC CT - #C CAT CTC CTG TCC AAT          480                                                                       Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Le - #u His Leu Leu Ser Asn                   145          - #       150          - #       155                      - - GAC ATG TGT GCT AGA GCT TAC TCT GAG AAG GT - #G ACA GAG TTC ATG TTG          528                                                                       Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys Va - #l Thr Glu Phe Met Leu               160              - #   165              - #   170                          - - TGT GCT GGG CTC TGG ACA GGT GGT AAA GAC AC - #T TGT GGG GGT GAT TCT          576                                                                       Cys Ala Gly Leu Trp Thr Gly Gly Lys Asp Th - #r Cys Gly Gly Asp Ser           175                 1 - #80                 1 - #85                 1 -      #90                                                                              - - GGG GGT CCA CTT GTC TGT AAT GGT GTG CTT CA - #A GGT ATC ACA TCA        TGG      624                                                                    Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gl - #n Gly Ile Thr Ser Trp                          195  - #               200  - #               205              - - GGC CCT GAG CCA TGT GCC CTG CCT GAA AAG CC - #T GCT GTG TAC ACC AAG          672                                                                       Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys Pr - #o Ala Val Tyr Thr Lys                       210      - #           215      - #           220                  - - GTG GTG CAT TAC CGG AAG TGG ATC AAG GAC AC - #C ATC GCA GCC AAC CCC          720                                                                       Val Val His Tyr Arg Lys Trp Ile Lys Asp Th - #r Ile Ala Ala Asn Pro                   225          - #       230          - #       235                      - - TGAGTGCCCC TGTCCCACCC CTACCTCTAG TAAACTGCAG     - #                      - #   760                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 238 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Ile Val Gly Gly Trp Glu Cys Glu Lys Hi - #s Ser Gln Pro Trp Gln        1               5 - #                 10 - #                 15              - - Val Ala Val Tyr Ser His Gly Trp Ala His Cy - #s Gly Gly Val Leu Val                   20     - #             25     - #             30                  - - His Pro Gln Trp Val Leu Thr Ala Ala His Cy - #s Leu Lys Lys Asn Ser               35         - #         40         - #         45                      - - Gln Val Trp Leu Gly Arg His Asn Leu Phe Gl - #u Pro Glu Asp Thr Gly           50             - #     55             - #     60                          - - Gln Arg Val Pro Val Ser His Ser Phe Pro Hi - #s Pro Leu Tyr Asn Met       65                 - # 70                 - # 75                 - # 80       - - Ser Leu Leu Lys His Gln Ser Leu Arg Pro As - #p Glu Asp Ser Ser His                       85 - #                 90 - #                 95              - - Asp Leu Met Leu Leu Arg Leu Ser Glu Pro Al - #a Lys Ile Thr Asp Val                  100      - #           105      - #           110                  - - Val Lys Val Leu Gly Leu Pro Thr Gln Glu Pr - #o Ala Leu Gly Thr Thr              115          - #       120          - #       125                      - - Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pr - #o Glu Glu Phe Leu Arg          130              - #   135              - #   140                          - - Pro Arg Ser Leu Gln Cys Val Ser Leu His Le - #u Leu Ser Asn Asp Met      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Ala Arg Ala Tyr Ser Glu Lys Val Thr Gl - #u Phe Met Leu Cys        Ala                                                                                             165  - #               170  - #               175             - - Gly Leu Trp Thr Gly Gly Lys Asp Thr Cys Gl - #y Gly Asp Ser Gly Gly                  180      - #           185      - #           190                  - - Pro Leu Val Cys Asn Gly Val Leu Gln Gly Il - #e Thr Ser Trp Gly Pro              195          - #       200          - #       205                      - - Glu Pro Cys Ala Leu Pro Glu Lys Pro Ala Va - #l Tyr Thr Lys Val Val          210              - #   215              - #   220                          - - His Tyr Arg Lys Trp Ile Lys Asp Thr Ile Al - #a Ala Asn Pro              225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 766 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..732                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GTG CCC CTC ATC CAG TCT CGG ATT GTG GGA GG - #C TGG GAG TGT GAG AAG           48                                                                       Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gl - #y Trp Glu Cys Glu Lys             1               5 - #                 10 - #                 15              - - CAT TCC CAA CCC TGG CAG GTG GCT GTG TAC AG - #T CAT GGA TGG GCA CAC           96                                                                       His Ser Gln Pro Trp Gln Val Ala Val Tyr Se - #r His Gly Trp Ala His                        20     - #             25     - #             30                  - - TGT GGG GGT GTC CTG GTG CAC CCC CAG TGG GT - #G CTC ACA GCT GCC CAT          144                                                                       Cys Gly Gly Val Leu Val His Pro Gln Trp Va - #l Leu Thr Ala Ala His                    35         - #         40         - #         45                      - - TGC CTA AAG AAG AAT AGC CAG GTC TGG CTG GG - #T CGG CAC AAC CTG TTT          192                                                                       Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gl - #y Arg His Asn Leu Phe                50             - #     55             - #     60                          - - GAG CCT GAA GAC ACA GGC CAG AGG GTC CCT GT - #C AGC CAC AGC TTC CCA          240                                                                       Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Va - #l Ser His Ser Phe Pro            65                 - # 70                 - # 75                 - # 80       - - CAC CCG CTC TAC AAT ATG AGC CTT CTG AAG CA - #T CAA AGC CTT AGA CCA          288                                                                       His Pro Leu Tyr Asn Met Ser Leu Leu Lys Hi - #s Gln Ser Leu Arg Pro                            85 - #                 90 - #                 95              - - GAT GAA GAC TCC AGC CAT GAC CTC ATG CTG CT - #C CGC CTG TCA GAG CCT          336                                                                       Asp Glu Asp Ser Ser His Asp Leu Met Leu Le - #u Arg Leu Ser Glu Pro                       100      - #           105      - #           110                  - - GCC AAG ATC ACA GAT GTT GTG AAG GTC CTG GG - #C CTG CCC ACC CAG GAG          384                                                                       Ala Lys Ile Thr Asp Val Val Lys Val Leu Gl - #y Leu Pro Thr Gln Glu                   115          - #       120          - #       125                      - - CCA GCA CTG GGG ACC ACC TGC TAC GCC TCA GG - #C TGG GGC AGC ATC GAA          432                                                                       Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gl - #y Trp Gly Ser Ile Glu               130              - #   135              - #   140                          - - CCA GAG GAG TTC TTG CGC CCC AGG AGT CTT CA - #G TGT GTG AGC CTC CAT          480                                                                       Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gl - #n Cys Val Ser Leu His           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - CTC CTG TCC AAT GAC ATG TGT GCT AGA GCT TA - #C TCT GAG AAG GTG        ACA      528                                                                    Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Ty - #r Ser Glu Lys Val Thr                          165  - #               170  - #               175              - - GAG TTC ATG TTG TGT GCT GGG CTC TGG ACA GG - #T GGT AAA GAC ACT TGT          576                                                                       Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gl - #y Gly Lys Asp Thr Cys                       180      - #           185      - #           190                  - - GGG GGT GAT TCT GGG GGT CCA CTT GTC TGT AA - #T GGT GTG CTT CAA GGT          624                                                                       Gly Gly Asp Ser Gly Gly Pro Leu Val Cys As - #n Gly Val Leu Gln Gly                   195          - #       200          - #       205                      - - ATC ACA TCA TGG GGC CCT GAG CCA TGT GCC CT - #G CCT GAA AAG CCT GCT          672                                                                       Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Le - #u Pro Glu Lys Pro Ala               210              - #   215              - #   220                          - - GTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TG - #G ATC AAG GAC ACC ATC          720                                                                       Val Tyr Thr Lys Val Val His Tyr Arg Lys Tr - #p Ile Lys Asp Thr Ile           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GCA GCC AAC CCC TGAGTGCCCC TGTCCCACCC CTACCTCTAG TA - #AA                    766                                                                      Ala Ala Asn Pro                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 244 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gl - #y Trp Glu Cys Glu Lys        1               5 - #                 10 - #                 15              - - His Ser Gln Pro Trp Gln Val Ala Val Tyr Se - #r His Gly Trp Ala His                   20     - #             25     - #             30                  - - Cys Gly Gly Val Leu Val His Pro Gln Trp Va - #l Leu Thr Ala Ala His               35         - #         40         - #         45                      - - Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gl - #y Arg His Asn Leu Phe           50             - #     55             - #     60                          - - Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Va - #l Ser His Ser Phe Pro       65                 - # 70                 - # 75                 - # 80       - - His Pro Leu Tyr Asn Met Ser Leu Leu Lys Hi - #s Gln Ser Leu Arg Pro                       85 - #                 90 - #                 95              - - Asp Glu Asp Ser Ser His Asp Leu Met Leu Le - #u Arg Leu Ser Glu Pro                  100      - #           105      - #           110                  - - Ala Lys Ile Thr Asp Val Val Lys Val Leu Gl - #y Leu Pro Thr Gln Glu              115          - #       120          - #       125                      - - Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gl - #y Trp Gly Ser Ile Glu          130              - #   135              - #   140                          - - Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gl - #n Cys Val Ser Leu His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Ty - #r Ser Glu Lys Val        Thr                                                                                             165  - #               170  - #               175             - - Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gl - #y Gly Lys Asp Thr Cys                  180      - #           185      - #           190                  - - Gly Gly Asp Ser Gly Gly Pro Leu Val Cys As - #n Gly Val Leu Gln Gly              195          - #       200          - #       205                      - - Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Le - #u Pro Glu Lys Pro Ala          210              - #   215              - #   220                          - - Val Tyr Thr Lys Val Val His Tyr Arg Lys Tr - #p Ile Lys Asp Thr Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Ala Asn Pro                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - TATACATATG TGGGACCTGG TTCTCTCC         - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - ATATGGATCC TCAGGGGTTG GCTGCGATGG T        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Met Trp Asp Leu Val Leu Ser Ile Ala Leu                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Met Trp Phe Leu Val Leu Cys Leu Ala Leu                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Met Trp Val Pro Val Val Phe Leu Thr Leu                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 237 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Ile Val Gly Gly Trp Glu Cys Glu Lys His Se - #r Gln Pro Trp Gln Val      1               5   - #                10  - #                15               - - Ala Val Tyr Ser His Gly Trp Ala His Cys Gl - #y Gly Val Leu Val His                  20      - #            25      - #            30                   - - Pro Gln Trp Val Leu Thr Ala Ala His Cys Le - #u Lys Lys Asn Ser Gln              35          - #        40          - #        45                       - - Val Trp Leu Gly Arg His Asn Leu Phe Glu Pr - #o Glu Asp Thr Gly Gln          50              - #    55              - #    60                           - - Arg Val Pro Val Ser His Ser Phe Pro His Pr - #o Leu Tyr Asn Met Ser      65                  - #70                  - #75                  - #80        - - Leu Leu Lys His Gln Ser Leu Arg Pro Asp Gl - #u Asp Ser Ser His Asp                      85  - #                90  - #                95               - - Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Ly - #s Ile Thr Asp Val Val                  100      - #           105      - #           110                  - - Lys Val Leu Gly Leu Pro Thr Gln Glu Pro Al - #a Leu Gly Thr Thr Cys              115          - #       120          - #       125                      - - Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Gl - #u Glu Phe Leu Arg Pro          130              - #   135              - #   140                          - - Arg Ser Leu Gln Cys Val Ser Leu His Leu Le - #u Ser Asn Asp Met Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Arg Ala Tyr Ser Glu Lys Val Thr Glu Ph - #e Met Leu Cys Ala        Gly                                                                                             165  - #               170  - #               175             - - Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gl - #y Asp Ser Gly Gly Pro                  180      - #           185      - #           190                  - - Leu Val Cys Asn Gly Val Leu Gln Gly Ile Th - #r Ser Trp Gly Pro Glu              195          - #       200          - #       205                      - - Pro Cys Ala Leu Pro Glu Lys Pro Ala Val Ty - #r Thr Lys Val Val His          210              - #   215              - #   220                          - - Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Al - #a Asn Pro                  225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - ATATGGATCC ATATGTCAGC ATGTGGGACC TGGTTCTCTC CA    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Val Pro Leu Ile Gln Ser Arg Ile Val Gly Gl - #y Trp Glu                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 261 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Met Trp Asp Leu Val Leu Ser Ile Ala Leu Se - #r Val Gly Cys Thr Gly        1               5 - #                 10 - #                 15              - - Ala Val Pro Leu Ile Gln Ser Arg Ile Val Gl - #y Gly Trp Glu Cys Glu                   20     - #             25     - #             30                  - - Lys His Ser Gln Pro Trp Gln Val Ala Val Ty - #r Ser His Gly Trp Ala               35         - #         40         - #         45                      - - His Cys Gly Gly Val Leu Val His Pro Gln Tr - #p Val Leu Thr Ala Ala           50             - #     55             - #     60                          - - His Cys Leu Lys Lys Asn Ser Gln Val Trp Le - #u Gly Arg His Asn Leu       65                 - # 70                 - # 75                 - # 80       - - Phe Glu Pro Glu Asp Thr Gly Gln Arg Val Pr - #o Val Ser His Ser Phe                       85 - #                 90 - #                 95              - - Pro His Pro Leu Tyr Asn Met Ser Leu Leu Ly - #s His Gln Ser Leu Arg                  100      - #           105      - #           110                  - - Pro Asp Glu Asp Ser Ser His Asp Leu Met Le - #u Leu Arg Leu Ser Glu              115          - #       120          - #       125                      - - Pro Ala Lys Ile Thr Asp Val Val Lys Val Le - #u Gly Leu Pro Thr Gln          130              - #   135              - #   140                          - - Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Se - #r Gly Trp Gly Ser Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Le - #u Gln Cys Val Ser        Leu                                                                                             165  - #               170  - #               175             - - His Leu Leu Ser Asn Asp Met Cys Ala Arg Al - #a Tyr Ser Glu Lys Val                  180      - #           185      - #           190                  - - Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Th - #r Gly Gly Lys Asp Thr              195          - #       200          - #       205                      - - Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cy - #s Asn Gly Val Leu Gln          210              - #   215              - #   220                          - - Gly Ile Thr Ser Trp Gly Pro Glu Pro Cys Al - #a Leu Pro Glu Lys Pro      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Val Tyr Thr Lys Val Val His Tyr Arg Ly - #s Trp Ile Lys Asp        Thr                                                                                             245  - #               250  - #               255             - - Ile Ala Ala Asn Pro                                                                  260                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 832 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 10..792                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - GGATCCAGC ATG TGG GAC CTG GTT CTC TCC ATC GCC - # TTG TCT GTG GGG             48                                                                                 Met Trp Asp Leu Val - #Leu Ser Ile Ala Leu Ser Val Gly                          1      - #         5         - #         10                        - - TGC ACT GGT GCC GTG CCC CTC ATC CAG TCT CG - #G ATT GTG GGA GGC TGG           96                                                                       Cys Thr Gly Ala Val Pro Leu Ile Gln Ser Ar - #g Ile Val Gly Gly Trp                15             - #     20             - #     25                          - - GAG TGT GAG AAG CAT TCC CAA CCC TGG CAG GT - #G GCT GTG TAC AGT CAT          144                                                                       Glu Cys Glu Lys His Ser Gln Pro Trp Gln Va - #l Ala Val Tyr Ser His            30                 - # 35                 - # 40                 - # 45       - - GGA TGG GCA CAC TGT GGG GGT GTC CTG GTG CA - #C CCC CAG TGG GTG CTC          192                                                                       Gly Trp Ala His Cys Gly Gly Val Leu Val Hi - #s Pro Gln Trp Val Leu                            50 - #                 55 - #                 60              - - ACA GCT GCC CAT TGC CTA AAG AAG AAT AGC CA - #G GTC TGG CTG GGT CGG          240                                                                       Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gl - #n Val Trp Leu Gly Arg                        65     - #             70     - #             75                  - - CAC AAC CTG TTT GAG CCT GAA GAC ACA GGC CA - #G AGG GTC CCT GTC AGC          288                                                                       His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gl - #n Arg Val Pro Val Ser                    80         - #         85         - #         90                      - - CAC AGC TTC CCA CAC CCG CTC TAC AAT ATG AG - #C CTT CTG AAG CAT CAA          336                                                                       His Ser Phe Pro His Pro Leu Tyr Asn Met Se - #r Leu Leu Lys His Gln                95             - #    100             - #    105                          - - AGC CTT AGA CCA GAT GAA GAC TCC AGC CAT GA - #C CTC ATG CTG CTC CGC          384                                                                       Ser Leu Arg Pro Asp Glu Asp Ser Ser His As - #p Leu Met Leu Leu Arg           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - CTG TCA GAG CCT GCC AAG ATC ACA GAT GTT GT - #G AAG GTC CTG GGC        CTG      432                                                                    Leu Ser Glu Pro Ala Lys Ile Thr Asp Val Va - #l Lys Val Leu Gly Leu                          130  - #               135  - #               140              - - CCC ACC CAG GAG CCA GCA CTG GGG ACC ACC TG - #C TAC GCC TCA GGC TGG          480                                                                       Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cy - #s Tyr Ala Ser Gly Trp                       145      - #           150      - #           155                  - - GGC AGC ATC GAA CCA GAG GAG TTC TTG CGC CC - #C AGG AGT CTT CAG TGT          528                                                                       Gly Ser Ile Glu Pro Glu Glu Phe Leu Arg Pr - #o Arg Ser Leu Gln Cys                   160          - #       165          - #       170                      - - GTG AGC CTC CAT CTC CTG TCC AAT GAC ATG TG - #T GCT AGA GCT TAC TCT          576                                                                       Val Ser Leu His Leu Leu Ser Asn Asp Met Cy - #s Ala Arg Ala Tyr Ser               175              - #   180              - #   185                          - - GAG AAG GTG ACA GAG TTC ATG TTG TGT GCT GG - #G CTC TGG ACA GGT GGT          624                                                                       Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gl - #y Leu Trp Thr Gly Gly           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - AAA GAC ACT TGT GGG GGT GAT TCT GGG GGT CC - #A CTT GTC TGT AAT        GGT      672                                                                    Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pr - #o Leu Val Cys Asn Gly                          210  - #               215  - #               220              - - GTG CTT CAA GGT ATC ACA TCA TGG GGC CCT GA - #G CCA TGT GCC CTG CCT          720                                                                       Val Leu Gln Gly Ile Thr Ser Trp Gly Pro Gl - #u Pro Cys Ala Leu Pro                       225      - #           230      - #           235                  - - GAA AAG CCT GTT GTG TAC ACC AAG GTG GTG CA - #T TAC CGG AAG TGG ATC          768                                                                       Glu Lys Pro Val Val Tyr Thr Lys Val Val Hi - #s Tyr Arg Lys Trp Ile                   240          - #       245          - #       250                      - - AAG GAC ACC ATC GCA GCC AAC CCC TGAGTGCCCC TG - #TCCCACCC CTACCTCTAG         822                                                                       Lys Asp Thr Ile Ala Ala Asn Pro                                                   255              - #   260                                                 - - TAAACTGCAG                - #                  - #                      - #       832                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 237 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Ile Val Gly Gly Trp Glu Cys Glu Lys His Se - #r Gln Pro Trp Gln Val      1               5   - #                10  - #                15               - - Ala Val Tyr Ser His Gly Trp Ala His Cys Gl - #y Gly Val Leu Val His                  20      - #            25      - #            30                   - - Pro Gln Trp Val Leu Thr Ala Ala His Cys Le - #u Lys Lys Asn Ser Gln              35          - #        40          - #        45                       - - Val Trp Leu Gly Arg His Asn Leu Phe Glu Pr - #o Glu Asp Thr Gly Gln          50              - #    55              - #    60                           - - Arg Val Pro Val Ser His Ser Phe Pro His Pr - #o Leu Tyr Asn Met Ser      65                  - #70                  - #75                  - #80        - - Leu Leu Lys His Gln Ser Leu Arg Pro Asp Gl - #u Asp Ser Ser His Asp                      85  - #                90  - #                95               - - Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Ly - #s Ile Thr Asp Val Val                  100      - #           105      - #           110                  - - Lys Val Leu Gly Leu Pro Thr Gln Glu Pro Al - #a Leu Gly Thr Thr Cys              115          - #       120          - #       125                      - - Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Gl - #u Glu Phe Leu Arg Pro          130              - #   135              - #   140                          - - Arg Ser Leu Gln Cys Val Ser Leu His Leu Le - #u Ser Asn Asp Met Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Arg Ala Tyr Ser Glu Lys Val Thr Glu Ph - #e Met Leu Cys Ala        Gly                                                                                             165  - #               170  - #               175             - - Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gl - #y Asp Ser Gly Gly Pro                  180      - #           185      - #           190                  - - Leu Val Cys Asn Gly Val Leu Gln Gly Ile Th - #r Ser Trp Gly Pro Glu              195          - #       200          - #       205                      - - Pro Cys Ala Leu Pro Glu Lys Pro Val Val Ty - #r Thr Lys Val Val His          210              - #   215              - #   220                          - - Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Al - #a Asn Pro                  225                 2 - #30                 2 - #35                          __________________________________________________________________________

What is claimed is:
 1. Isolated, substantially homogenous pre-pro hK2polypeptide comprising an amino acid sequence corresponding to SEQ IDNO:19.
 2. An isolated variant hK2 polypeptide wherein the variant hK2polypeptide has 100% identity to SEQ ID NO:16 at amino acid positions15-26, 41-56 and 153-167 of SEQ ID NO:16, wherein said polypeptide bindsto an antibody that binds to mature hK2 but not to PSA, wherein at leastone amino acid at position 1-14, 27-40, 57-152 or 168-237 of SEQ IDNO:16 is substituted relative to SEQ ID NO:16.
 3. The isolated hK2polypeptide of claim 2 wherein the substitution is a conservativesubstitution.
 4. Isolated, substantially homogenous pro or mature hK2polypeptide comprising an amino acid sequence corresponding to SEQ IDNO:21.